1. Field of the Invention
The present invention relates to computer-implemented methods and computer readable storage media containing programs for organizing image data produced by resonant Mie Scattering of light from microparticles disposed in a free array, and to the resulting data structures produced therefrom.
2. Description of Related Art
Copending application WO 2004/044232, filed Nov. 6, 2003, Prober et al., titled “Microparticle-based Methods and Systems and Applications Thereof”, assigned to the assignee of the present invention, discloses various methods, systems and applications that employ resonant light scattering as an analytical tool for determining a microparticle's identity as well as the presence and, optionally, the concentration, of one or more target analytes on the microparticle.
In general, a high refractive index microparticle is irradiated with light of a given wavelength and the microparticle scatters a portion of that light to a detector. As the incident wavelength is scanned, i.e., varied over an analytical wavelength range, a pattern or spectrum of scattered light as a function of wavelength results. The resonant light scattering spectra is detected by an imaging detector, such as a charge-coupled device (CCD) camera. The camera produces an electronic image of the particles at each wavelength step.
Thus, a complete wavelength scan results in a series of digital images of all the microparticle, one image for each wavelength interval in the scan. The intensity of scattering from a microparticle at a given wavelength is related to the brightness of that microparticle's scattering image at that wavelength. Computerized image processing and spectral analysis are required to obtain the complete scattering spectra of all the particles from the set of images obtained during the scan. Each particle has a distinct resonance light scattering pattern, due to natural processing variations, that can be used to identify the particle. The presence and optionally the concentration of a target analyte can be determined from the shift in the resonance light scattering pattern that occurs when the analyte binds to a capture probe immobilized on the surface of the particle. The magnitude of the shift is related to the concentration of the analyte in the solution.
Shown in FIG. 1 is a diagrammatic illustration of an image detection apparatus used to create images of scattered light from a plurality of microparticles 2 called a “free array”. A population of the microparticles is isolated in an imaging optical cell 19 and, using a scanning diode laser 22 as a light source, imaged with a microscope 21. The microparticles could be randomly distributed or distributed in an ordered fashion such as in a tube or in a linear or rectangular array by constraining them in grooves, channels, or indentations on a substrate. The magnification is set to simultaneously image the particles of interest.
As the laser wavelength is increased through the wavelength range the microparticles are illuminated and a digital image is produced at each of a predetermined number (“Q”) of wavelengths. The light scattered from microparticles in the field is collected and imaged by the objective lens 20 and associated optics of a camera 26. Illuminating wavelengths in the range of from about 770 nanometers to about 780 nanometers are particularly useful for the images under discussion. For a ten (10) nanometer range, a scan having using an illuminating laser having a “slew rate” (wavelength step, in nanometers per second) on the order of 0.2 results in a scan having a length of fifty (50) seconds. At an imaging rate on the order of thirty (30) images per second an experimental scan can produce about fifteen hundred images (Q=1500).
The camera 26 could be any imaging device capable of the speed and sensitivity required for this application. The camera 26 is preferably a digital camera having an electronic image plane based on a two-dimensional CCD (charge coupled device) or equivalent imaging means. The camera 26 thus produces an electronic image of the particles at each wavelength step (e.g., 0.2 nanometers per second). Preferably, the camera functions and data acquisition are controlled by a computer 10 operably linked to the camera 26 and to the scanning light source. The computer has software suitable for these purposes.
Each of the Q images produced by the digital camera 26 may be viewed on a video monitor 17 connected to the camera 26. Simultaneously, each image is captured by a suitable image capture card or other device 27 that functions to create (after appropriate analog-to-digital conversion) a digital representation of the electronic image on the electronic image plane of the camera. The digital image is stored in memory of the computer 10. A suitable image capture card is that sold by National Instruments Corporation as an IMAQ PCI 1428 image capture card.
A typical visual image taken at one given wavelength of polarized light from such a set of Q images is shown in FIG. 2. Light scattered by the mechanism of resonant Mie scattering emanates along the rim of a microparticle in the form of a substantially continuous (yet segmented) substantially circular rings. The incident and scattered light beams used to produce the visual and diagrammatic images of FIG. 2 were polarized independently, with the two axes of polarization parallel to each other. This results in sectors of scattered light centered approximately at the 12:00, 3:00, 6:00, and 9:00 positions of the substantially circular microparticle images.
As noted the intensity of resonant Mie scattering from a microparticle a given wavelength is related to the brightness of that particle's scattering image at that wavelength. This may be most graphically understood from FIGS. 3A through 3F, which are a series of stylized diagrammatic renderings of the visual image of FIG. 2 at respective illuminating wavelengths L1 through LQ. FIGS. 4A through 4F are a series of diagrams representing the images on the electronic image plane corresponding to each of the visual images. In FIGS. 4A through 4F the relative intensity values of the pixels are indicated by decimal numeric values.
As seen from close inspection of the renderings shown in FIGS. 3A through 3F and FIGS. 4A through 4F the intensity of the light scattered from the same given portion of the same microparticle changes over the wavelength scan. These intensity differences define a spectrum of scattered light as a function of wavelength for various locations on the microparticle. By monitoring changes in spectra over time and/or in the presence and optionally the concentration of a target analyte be obtained.
Each image of an entire viewed field (FIG. 2) or image of any selected region of interest therein (e.g., that portion bounded by the white border in FIG. 2) is typically in the shape of a rectangular or square matrix, or grid, as suggested in FIGS. 3 and 4. It should be appreciated, however, that the pixels in the image could be arranged to form other shapes on the image plane, such as a continuous or segmented annulus. Whatever its shape, the image contains a predetermined number (“N”) of pixels.
For example, in a square image having length and width dimensions on the order of one thousand by a thousand (1000×1000) pixels, the total number N of pixels in an image (whether the “image” is defined as the entire field or a selected region of interest of a field) could be on the order of one million (1,000,000). When one considers that in a typical experimental scan approximately fifteen hundred images may be produced (i.e., Q=1500) the data storage requirements (assuming at least ten-bit digital accuracy) for the complete set of such digital representations of the Q images from this single experiment could be on the order of three (3) gigabytes.
Handling this prodigious amount of data and retrieving selected portions of this data for processing purposes (e.g., for effecting analysis of the microparticles) is problematic. In addition, the mere storage of such a vast amount of data presents issues of considerable difficulty.
Accordingly, in view of the foregoing it is believed advantageous to provide methods and programs for organizing the image data produced by the scattering of light (particularly resonant Mie scattering) from microparticles disposed in a free array that make the handling and retrieval of information for display, analysis or other purposes more rapid and more efficient. It is believed to be of further advantage if such data organization methods result in a more compact and manageable amounts of data, thus reducing significantly the data storage requirements for free array testing.